Using simple retroviruses as a model system, we have examined three aspects of virus assembly. We have dissected a motif that is important for virus assembly and located at the C-terminal region of murine leukemia virus (MLV) capsid (CA). From data generated in our mutational analyses, we hypothesize that the sequences in this motif form an &#945;-helix; maintenance of the helical structure and the phase of the helix are critical to its function. We are performing experiments to further investigate this motif to gain insight into the functional domains of CA in virus assembly. We have performed experiments to determine the domain(s) in Gag that is important for the coassembly of MLV and spleen necrosis virus (SNV) Gag. MLV and SNV are distantly related viruses. Using hybrid viruses, we determined that homologous CA is needed for functional complementation of MLV and SNV Gag. We have also studied the MLV Gag components that are important for early virus replication events. We found that homologous CA and p12 domains are required for efficient virus infection. This study indicated that the mature CA and p12 proteins cooperate during early MLV replication, most likely for the transport of the reverse transcription/preintegration complex. We have also examined the effects of Gag mutations on virion RNA dimer maturation. We examined two Gag mutants: an MLV CA deletion mutation and an HIV-1 PTAP substitution mutant. We found that although most of Gag proteins from these virions were cleaved, virion RNA dimers did not mature. These data suggest that virion RNA dimer maturation requires more than proteolytic cleavage of Gag and is likely to be associated with virion morphological changes. We are now studying functional complementation and coassembly in lentivirus-based systems namely, HIV-1 and HIV-2. Using a genetic complementation assay and an imaging approach, we have demonstrated that HIV-1 and HIV-2 Gag can coassemble; furthermore, these coassembled viruses can be infectious. To our knowledge, this is the first study of its kind to demonstrate that two lentiviruses can have such interactions. We are currently following several leads in this study. We intend to define the extent of heterologous Gag interaction, and determine the advantage of such mixed particles in virus replication. For example, we intend to determine whether mixed particles can better counter drug treatment and adapt to replication of a different host cell via bypassing of the cellular defense proteins. Additionally, we are studying the RNA packaging mechanisms of HIV-1 and other lentiviruses. Retroviruses can interact with each other through complementation. Proteins and RNAs from two different viruses can comingle and interact in doubly infected cells. An estimated one million people are dually infected with HIV-1 and HIV-2, providing the basis for possible interactions between these two viruses in human populations. Using two different systems, we examined whether heterologous Gag proteins can coassemble and complement each others functions. We have observed that MLV and SNV Gag can interact but their genomes must encode a homologous capsid (CA) domain in Gag to rescue virus replication. In contrast, the Gag proteins of HIV-1 and HIV-2 can coassemble and rescue virus replication via functional complementation. We will study the properties of particles containing both HIV-1 and HIV-2 Gag, and further define the extent of interactions and complementation of Gag proteins from different retroviruses. We will quantify the functional nucleocapsid (NC) and late-domain motif needed for virus replication and will determine the requirement of various viral protein functions in different steps of viral replication. With our newly developed imaging systems, we will study the Gag-Gag and Gag-RNA interactions in living cells. We will also explore the link between formation of the condensed core and a stable RNA dimer. Results from these studies will provide insights into retroviral interactions via complementation and will further our understanding of the process of virus assembly and the functions of Gag proteins in various stages of viral replication. [Corresponds to Hu Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program